Nucleosome Dynamics and the Repair of DNA Damage

描述(由申请人提供)：DNA损伤的修复对于保护基因组的完整性和防止可能导致癌症的遗传毒性事件至关重要。然而，哺乳动物细胞含有一系列不同的功能和结构染色质结构域，这些结构域在组蛋白修饰模式、染色质结合蛋白和核小体堆积密度方面都不同。因此，损伤部位染色质结构的重塑对于DNA损伤的检测和修复至关重要。然而，驱动DSB核小体结构变化的潜在机制仍然不清楚。我们的初步数据表明，组蛋白变异体组蛋白H2A.Z在DNA双链断裂(DSB)时迅速交换到核小体上。H_2A.Z在DSB上的交换改变了核小体的动态，推动了开放的、灵活的染色质结构域的形成。此外，这种H2A.Z交换促进了组蛋白修饰的特定模式，对于控制DNA在断裂位置的切除和处理是至关重要的。中心假设是，H2A.Z交换驱动DSB染色质的重塑，并控制组蛋白修饰和DNA的末端处理。锌指核酸酶(ZFN)文库将被用来在活跃转录的基因和紧凑的基因间隔区创建序列特定的DSB。我们将确定H2A.Z如何在基因和基因间隔区交换重塑染色质结构，并确定这些不同染色质区域之间修复机制的关键差异。我们将确定H2A.Z交换如何影响核小体在DSB的定位，并确定核小体在DSB的定位如何影响随后组蛋白的修饰和末端切除的程度。此外，我们将确定H2 A.Z上的关键结构域，这些结构域改变核小体动力学并促进DSB上开放染色质结构的形成。此外，我们还将确定DSB上H_2A.Z-核小体的存在如何影响DSB修复的机制和保真度。通过使用锌指核酸酶在基因和基因间隔区创建DSB，我们可以确定DSB修复是如何在不同的功能区域进行的，并揭示H2A.Z在改变DSB局部染色质结构中的重要性。此外，由于许多肿瘤细胞既改变了染色质结构，又增加了组蛋白H2A.Z的水平，这项工作将为染色质结构和组蛋白H2A.Z如何影响与癌症发生、肿瘤进展以及肿瘤对放射治疗和化疗的敏感性相关的过程提供新的见解。

DESCRIPTION (provided by applicant): The repair of DNA damage is critical to protect the integrity of the genome and prevent genotoxic events which can lead to cancer. However, mammalian cells contain a diverse array of functional and structural chromatin domains, which differ in the pattern of histone modifications, chromatin binding proteins and in the density of nucleosome packing. Consequently, remodeling of the chromatin structure at sites of damage is critical for the detection and repair of DNA damage. However, the underlying mechanism driving changes in nucleosome structure at DSBs remains poorly defined. Our preliminary data demonstrates that a histone variant, histone H2A.Z, is rapidly exchanged onto nucleosomes at DNA double-strand breaks (DSBs). The exchange of H2A.Z at DSBs alters nucleosomes dynamics, driving the formation of open, flexible chromatin domains at the break. Further, this H2A.Z exchange promotes specific patterns of histone modification and is critical for controlling resection and processing of the DNA at the break site. The central hypothesis is that H2A.Z exchange drives remodeling of the chromatin at DSBs and controls both histone modification and end processing of the DNA. A library of Zinc Finger Nucleases (ZFNs) will be used to create sequence-specific DSBs in actively transcribed genes and compact, intergenic regions. We will determine how H2A.Z exchange remodels chromatin structure in genes and intergenic regions and identify key differences in the mechanism of repair between these distinct chromatin domains. We will determine how H2A.Z exchange impacts the positioning of nucleosomes at DSBs and determine how nucleosome positioning at DSBs impacts the subsequent modification of histones and the extent of end resection. Further, we will identify key domains on H2A.Z which alter nucleosome dynamics and promote the formation of open chromatin structures at DSBs. In addition, we will determine how the presence of H2A.Z-nucleosomes at DSBs impacts the mechanism and fidelity of DSB repair. By using Zinc Finger Nucleases to create DSBs in genes and intergenic regions, we can determine how DSB repair proceeds in distinct functional domains and unravel the importance of H2A.Z in altering the local chromatin architecture at DSBs. Further, because many tumor cells have both altered chromatin organization and increased levels of histone H2A.Z, this work will provide new insights into how chromatin structure and H2A.Z impacts processes related to carcinogenesis, tumor progression and the sensitivity of tumors to both radiation therapy and chemotherapy.